Volume 74, Issue 8, Pages 1085-1093 (October 2008) The impact of sodium chloride and volume depletion in the chronic kidney disease of congenital chloride diarrhea  Satu Wedenoja, Timo Örmälä, Ulla B. Berg, Stella F. Edström Halling, Hannu Jalanko, Riitta Karikoski, Juha Kere, Christer Holmberg, Pia Höglund  Kidney International  Volume 74, Issue 8, Pages 1085-1093 (October 2008) DOI: 10.1038/ki.2008.401 Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 1 Renal injury in cases with CLD. (a) Native kidney biopsy specimen from case 1, taken at age 19, shows a sclerosed glomerulus and abundant von Kossa-positive (black) tubular calcifications, which are (b) nonpolarizable, that is, have the same optical properties in all directions under polarized light. (c) Kidney biopsy specimen from case 1, taken 3 years after transplantation, demonstrates large von Kossa-positive (black) tubular calcifications and single interstitial calcium deposits. (d) The removed first transplant of case 2 shows moderate tubular atrophy, luminal dilation, and abundant von Kossa-positive (black) tubular calcifications located in the cortical distal nephrons, whereas (e) the proximal tubules detected with angiotensin-converting enzyme (ACE) (brown) contain no calcifications. (f) Higher magnification from the same specimen exemplifies cortical calcification both in the distal tubules and, less abundantly, in the surrounding interstitium. (g) Polarization microscopy reveals the nonpolarizable nature of these calcificatioms. (h) Kidney biopsy specimen from case 2, taken 8 weeks after the second transplantation, shows von Kossa-positive (black) tubular calcifications located in the distal tubules. (i) In the consecutive section, these tubules (arrows) differ from the proximal tubules stained with ACE (brown). Stainings: hematoxylin and eosin (b, f, g), von Kossa (a, c, d, h), and ACE (e, i). Magnifications: × 10 (d, e), and × 20 (other micrographs). Kidney International 2008 74, 1085-1093DOI: (10.1038/ki.2008.401) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 2 Heights, renal lengths and glomerular filtration rates. (a) Standard deviations (s.d.) for heights (•) were significantly higher than those of renal lengths (○) in 35 patients with congenital chloride diarrhea (CLD). Calculated median values were 0 s.d. for heights and -1.1 s.d. for renal lengths. (b) Relative renal lengths showed a positive correlation with glomerular filtration rates (GFR). Kidney International 2008 74, 1085-1093DOI: (10.1038/ki.2008.401) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 3 Glomerular filtration rate, urinary electrolytes, blood pressures, and erythrocyte sedimentation rates. Excretion of (a) urinary Cl− and (b) Na+, and (c) systolic blood pressure (BP) were significantly lower and (d) erythrocyte sedimentation rates (ESRs) significantly higher among the subgroup of 20/35 patients with congenital chloride diarrhea (CLD) and glomerular filtration rate (GFR) below 110ml/min per 1.73m2. Data are presented as means and 95% confidence intervals. Urinary excretion of Ca2+ showed a positive correlation with both (e) urinary excretion Cl− and (f) Na+ in 35 patients with CLD. Kidney International 2008 74, 1085-1093DOI: (10.1038/ki.2008.401) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 4 Urinary NaCl and pH, vein blood HCO3−, and serum aldosterones. (a) Urinary excretion of Na+ was significantly lower and (b) urinary pH higher among the subgroup of 15/35 patients with congenital chloride diarrhea (CLD) and urinary Cl− excretion below 85mmol/day. No differences emerged in (c) vein blood HCO3− or (d) serum aldosterone levels. Data are presented as means and 95% confidence intervals. Abbreviation ns indicates nonsignifant P value. Kidney International 2008 74, 1085-1093DOI: (10.1038/ki.2008.401) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 5 SLC26A3 in human kidney. (a) Ethidium bromide staining showed variable levels of solute carrier family 26 member 3 (SLC26A3) mRNA, with the expected product size of 348bp, in 12 samples from human kidney cortex (kidney samples), expression being significantly higher in the positive control of colon, and absent in both the negative control of skeletal muscle (m), and in water control (w). The PCR products were purified and sequenced in both directions. (b) GAPDH controls for the 12 samples from the human kidney cortex. (c) SLC26A3 mRNA was detected also in two commercial cDNA samples from human kidney. (d) Western blotting showed very slight bands of ∼75kDa in four samples from human kidney cortex (1–4) and in one commercial sample (5) from human kidney, the positive control being colon, and negative control liver. (e) Apical staining for SLC26A3 (brown) was present only in a subset of cells of the connecting tubule (CNT) (upper positive tubule) and cortical collecting duct (CCD) (lower positive tubule), shown also (f) in higher magnification. (g) Negative control stained with the SLC26A3 preimmune serum. In the CCD of the medullary rays, (h) expression of SLC26A3 (brown) appeared apically in a few cells differing from (i) the principal cells positive for aquaporin 2 (AQP2) in the consecutive section (brown). (j) In the CNT, positive labeling for SLC26A3 (brown) was present in the cell type with mainly basal and cytoplasmic labeling for vacuolar type H+-ATPase (V-ATPase) (red). (k) Similarly, cells in the CCD with the apical signal (arrows) for SLC26A3 (brown) had mainly basal and cytoplasmic labeling for V-ATPase (red). Both the apical signal for (l) solute carrier family 26 member 4 (SLC26A4) and (m) SLC26A3 emerged in the CCD, one apical cell outline (arrow) even suggesting localization in the same cell in consecutive sections. The kidney sample of the patient with CLD showed (n) lack of expression of SLC26A3 but (o) positive signal for SLC26A4 (arrows) in the consecutive section. Magnifications: × 20 (e, g–i), × 40 (f), × 63 (l–o), and × 100 (j, k). Kidney International 2008 74, 1085-1093DOI: (10.1038/ki.2008.401) Copyright © 2008 International Society of Nephrology Terms and Conditions